Figure 1

The work flow for proteomic and bioinformatics PTM analysis is illustrated. [A] Proteins isolated from kinase assays are in-solution digested into peptides using the proteases Lysyl Endopeptidase and Trypsin. The peptides containing specific post-translational modifications (phosphorylation) are enriched using different resins. Non-modified peptides are used to identify proteins. [B] Purified peptides are separated on a miniaturized reverse phase chromatography column with an organic solvent gradient. Peptides eluting from the column are ionized by electrospray at the tip of the column, directly in front of the mass spectrometer. [C] The electrosprayed ions are transferred into the vacuum of the mass spectrometer. In the mass spectrometer (MS mode) all ions are moved to the mass analyzer (ion Trap), where they are measured at high resolution. The mass analyser then selects a particular peptide ion and fragments it in a collision cell. For modified peptides, the peptide mass will be shifted by the mass of the modification, as will all fragments containing the modification, allowing the unambiguous placement of the PTM on the sequence. [D] The mass and lists of fragment masses for each peptide are scanned against protein sequence databases, resulting in a list of identified peptides and proteins. The lists of proteins and their peptides are the basis for bioinformatics analysis, in order to acknowledge improvements.